Secreted Mac-2-binding glycoprotein

ABSTRACT

A purified glycoprotein complex of over 1200 kD apparent native molecular weight having a sedimentation value of approximately 25S and having the ability to selectively bind human Mac-2 or interfere with PHA activation of lymphocytes, DNA sequences that encode the protein, and expression systems for expressing it, thus providing for reagents that are useful for treating or diagnosing diseases, including cancer, infectious disease, and diseases of the immune system, and for monitoring the concentration of the glycoprotein in human milk.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 08/316,714,filed 29 Sep. 1994, which is a continuation of Ser. No. 07/961,404,filed 15 Oct. 1992, now abandoned which is a continuation-in-part ofSer. No. 07/777,121, filed 16 Oct. 1991, now abandoned.

FIELD OF THE INVENTION

This invention is in the area of molecular biology/biochemistry andpresents a purified glycoprotein that binds to the human lectin Mac-2,and would play an important role in interaction events at cell surfaces,DNA sequences that encode the protein, and expression systems forexpressing it. The protein has a variety of medical applicationsinvolving the regulation of cell surface interactions relating to immuneresponses, pathogen/host cell interactions, metastasis, and celladhesion and migration, and developmental functions such as pre-antibodyimmunity.

BACKGROUND OF THE INVENTION

Cell surface molecules play a key role in the infectivity of viruses andother pathogens with their target cells. For example, ICAM-1, theendothelial cell receptor for integrin binding, is also the receptor forrhinovirus binding. Rhinoviruses are members of the picornavirus familyand are responsible for about 50% of common colds in humans. Anotherclassic example of such interactions is influenza hemagglutinin, alectin that binds to sialic acid on its host cell as the first step ininfection. Thus, a prophylactic approach to preventing the common coldis to interfere with the binding of rhinovirus to cell-bound ICAM-1 byadministration of soluble binding competitors to the host receptor.

Lectins are a class of proteins that bind carbohydrates specifically andnoncovalently. Lis, H. and Sharon, N., 1986, Annual Review ofBiochemistry, 55:35. Numerous lectins have been identified in higheranimals, both membrane-bound and soluble, and have been implicated in avariety of Cell-recognition phenomena, in addition to roles that theyplay in metastasis.

Lectins may generally be classified as either of the C-type, whosebinding properties are calcium-dependent and which are structurallyrelated to the asialoglycoprotein receptor, or the S-type, orthiol-dependent lectin. It should be noted, however, that there areother proteins with lectin properties that apparently do not fall intoeither of these classes, such as for example, fibronectin and laminin.Drickamar, K., 1988, J. Biol. Chem., 263:9557.

Lectins are thought to play a role in regulating cellular events thatare initiated at the level of the plasma membrane. For instance, plasmamembrane associated molecules are involved in the activation of varioussubsets of lymphoid cells, particularly T-lymphocytes, and it is knownthat cell-surface molecules are responsible for activation of thesecells and consequently their response during an immune reaction. Thisphenomenon has been studied using various plant lectins, such asleucoagglutinating phytohaemagglutinin (PHA) and concanavalin A (con A).These molecules are thought to activate T-cells by binding tocarbohydrate moieties associated with specific molecules on theT-lymphocyte cell surface.

One known human lectin, originally described as a cell-associatedmacrophage antigen, is called "Mac-2". Ho & Springer, J. Immunol.,(1982) 128:1221-1228. The 32-kDa lectin Mac-2, is expressed atsignificant levels in thioglycolate-elicited mouse macrophages but notin resident macrophages and may be involved in cell adhesion or immuneresponses. The human homologue of Mac-2 has been cloned and shown to bea lactose/galactose-specific lectin that is externalized despite lackinga leader sequence. Oda et al., Gene (1991) 99:279-283; Cherayil et al.,PNAS (USA) (1990) 87:7324-7438. Mac-2 is identical or closely related toother previously described lectins: EBP, a protein believed to representa new type of cell adhesin (Frigeri & Liu, J. Immunol. (1992)148:861-867), CBP35, a galactose-binding lectin (Jai & Wang, J. Biol.Chem. (1988) 263:6009-6011), a non-integrin laminin-binding protein (Wooet al., J. Biol. Chem. (1990) 265:7097-7099), RL-29, a lactose-specificlung lectin (Leffler & Barondes, J. Biol. Chem. (1986) 261:10119-10126),and L-34, a galactose-binding lectin correlated with neoplastictransformation and metastasis (Raz et al., Int. J. Cancer (1990)46:871-877). Mac-2 is present in significant concentrations in the tipsof intestinal villi, where it may be a target for colonization by humanpathogens.

Several researchers have isolated and purified glycoproteins that act asligands for various lectins. For example, a protein termed thetamm-horsfall glycoprotein has been shown to inhibit lymphocyteactivation induced by several lectins, including leucoagglutinin andhemagglutinin from Phaseolus vulgaris. Serafini-Cessi, F. et al., 1979,Biochemical Journal, 183:381-388. Additionally, glycoproteins that actas PHA-binding factors have been partially purified from porcine spleniclymphocytes. Further studies also show that PHA activation of porcinelymphocytes is inhibited by the partially purified glycoproteins.Dupuis, G. et al., 1985, Canadian Journal of Biochemistry & CellBiology, 63:932-940. These researchers were unable to identify a precisemolecular weight species that exhibited the inhibitory activity. Rather,they reported a range of molecular weight species in partially purifiedpreparations, as revealed by Coomassie Blue staining of sodium dodecylsulphate polyacrylamide gels. Major bands were observed having apparentmolecular weights of about 50-55, 75, 95, 130, and 155 kD; additionalminor species exhibited apparent molecular weights of about 42, 45,60-65, 175, and 200-250 kD.

Similar studies by other investigators have shown the existence of otherPHA-binding molecules. For instance, a PHA-binding factor from pigmesenteric lymph nodes has been isolated and shown to have a molecularmass of about 100 kD. Allan, D. and Crumpton, N.J., 1973, Exp. CellRes., 78:271-278. A PHA-binding molecule present in plasma membranesfrom pig submaxillary lymph node lymphocytes was shown to exhibit anapparent molecular mass greater than 94 kD. Alexander, S. et al., 1978,Biochemical Biophys. Acta, 512:350-364. Other PHA-binding ligands havebeen isolated from human peripheral blood by affinity chromatography andhave been found to have molecular masses in the range of 20-35, 43, 60,and 70 kD. Skoog, B. et al., 1980, Scand. Journal Immun., 11:369-376.Other researchers have reported the presence of leucoagglutinin-receptorglycoproteins with molecular weights ranging from 43 to 250 kD inneuraminidase-treated peripheral human T lymphocytes.

The health benefits of human breast milk have long been recognized.Recently, the prophylactic effects of milk in preventinggastrointestinal infections have been described. Gerrard, J., 1974,Pediatrics, 54:757-764. At least in part, this is due tonon-immunoglobin glycoproteins present in milk that have bindingproperties that protect newborns from viral or bacterial infections.Lonnerdal, B., 1985, Am. J. Clin., 42:1299-1317 and Holmgren, J. et al.,1983, Infect. Immun., 33: 459-463. These proteins are thought to exerttheir effects, at least in part, by preventing or disrupting theadherence of bacteria or viruses to intestinal epithelium by binding tobacterial adhesins or viral hemagglutinins. Bacterial adhesins and viralhemagglutinins are surface molecules that facilitate the adherence ofthese organisms to epithelial cell surfaces as an early step ininfection. The milk proteins involved in this process have not been wellcharacterized. Holmgren, J. et al., 1983, Infect. Immun., 33:459-463.However, a glycoprotein having a molecular weight above 400,000 has beendescribed that is able to neutralize respiratory syncytiaI virus.Laegreid, A. et al., 1986, Acta Paediatric. Scand., 75:696-701. Inaddition to the immuno-protective and anti-infective functions providedby such proteins, other human milk proteins serve special roles ascarriers of specific nutrients.

Similarly, a high molecular weight material has been identified in humanserum that interferes with the attachment and infectivity of hepatitis Avirus to various cell lines. Zajac, A. et al., 1991, J. of Gen. Virol,72:1667-1675. This material has not been purified nor have itsproperties been further characterize&

Rosenberg et al., J. Biol. Chem. (1991) 266:18731-18736, describe aMac-2 binding protein from colon carcinoma cells, and report a partialN-terminal protein sequence. Linsley et al., Biochem. (1986)25:2978-2986 have characterized a lung carcinoma protein, L3, with asimilar, but not identical N-terminal protein sequence. No one has asyet identified the complete amino acid sequence for a novel glycoproteinspecific for binding to the human Mac-2 lectin, nor has the cDNAencoding such a sequence been cloned.

SUMMARY OF THE INVENTION

An object of the invention is the description of a substantiallypurified protein that binds to the Mac-2 lectin. This "Mac-2 bindingprotein" has an apparent native, molecular weight of over 1200 kD, asedimentation value of approximately 25S, and is composed ofglycosylated subunits having approximately 567 amino acids. Depending onthe source and degree of glycosylation, the apparent molecular weight ofthe unproteolyzed subunits is about 85-97 kD as revealed by SDS gelelectrophoresis.

A second object of the invention is the description of DNA sequencesthat encode the subunits, and vectors for expressing the protein encodedby the sequences.

A third object of the invention is the description of medicamentsconsisting of the lectin-binding protein that are useful in treating orpreventing diseases that result from binding of a disease-causing agentto the cell surface of a target cell.

A fourth object of the invention is the description of medicaldiagnostics, preferably antibody in nature, for determining theconcentration of gp85-97 protein in biological fluids, which informationcan be predictive of the health status of an individual including thesusceptibility of the individual to disease. A preferred application isin monitoring the concentration of gp85-97 protein in human milk todetermine if an infant is receiving the appropriate amount of thisprotein for maximum health benefit.

A fifth object of the invention is a composition consisting of infantformulas supplemented with gp85-97 protein, either recombinant or thepurified native protein, that can be fed to an infant in need of themedical benefit afforded by the protein.

A sixth object of the invention is the presentation of methods oftreating cancer, preferably breast cancer, consisting of administeringthe molecule in amounts effective to treat the cancer.

A seventh object of the invention is a method for treatment ofinfectious diseases in a patient, preferably those caused by pathogensthat colonize nasal passages or intestinal epithelium, consisting ofadministering to a patient in need of such treatment a therapeuticallyeffective amount of gp85-97 and a pharmaceutically acceptable carrier.

These and other objects of the invention will become apparent uponreading the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an analytical Phenyl-TSK HPLC chromatographic profile ofSK-BR-3 secreted gp97. Inhibition of PHA-stimulated cell proliferationactivity was assayed to reveal fractions that contained gp97.

FIG. 2 shows a sucrose gradient centrifugation profile of purifiedSK-BR-3 gp97 as detected using a non-denaturing dot-blot assay withanti-gp85-97 antibody. Analysis of a representative preparation ofpurified gp97 by SDS-PAGE is also shown.

FIG. 3 shows the apparent native molecular mass of gp97 isolated fromSK-BR-3 cells determined via chromatography on a Sepharose 6 sizeexclusion HPLC column.

FIG. 4 shows a Bio-Sil SEC 250 size exclusion-HPLC chromatographicprofile of partially purified SK-BR-3 gp97 digested or not digested withtrypsin.

FIG. 5 shows Western blot analysis of COS cell-expressed gp85-97.

FIG. 6 shows a Phenyl TSK HPLC chromatographic profile of gp85 fromhuman breast milk along with SDS-PAGE analysis of the peak fraction.

FIG. 7 shows a Western blot analysis of various human fluids withanti-gp85-97 antibody.

FIG. 8 shows a Western blot analysis of a co-precipitation of gp85-97from HT-29 and HL-60 cells.

Table 1 shows the effects of anti-gp85-97 antibody on ³ H-thymidineincorporation by SK-BR-3 cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein is related to previously published workand pending patent applications. By way of example, such work consistsof Scientific papers, patents Or pending patent applications. All ofthese publications and applications, cited previously or below arehereby incorporated by reference in their entirety.

As used herein, "gp85-97" is defined to mean a class of structurallyhomologous molecules having lectin-binding properties and biologicalactivities described below. "Structurally homologous" is defined to meanproteins containing substantially identical polypeptide backbones,encoded by the gp85-97 gene and proteolytically processed by cellularenzymes to generate a "mature" N-terminus by removal of the leadersequence. This form of the protein may or may not be furtherproteolytically nicked, and its amino acid side chains and/orglycosylation sites may also be modified to various extents. The precisechemical structure of gp85-97 depends on a number of factors. Twomembers of the class are defined as "gp97" and "gp85", which have beenisolated from SK-BR-3 cell culture supernatant fluid and human milk,respectively. In their native forms the subunits present in "gp85-97"are understood to occur in a native "complex" of over 1200 kD apparentmolecular weight and having a sucrose velocity gradient sedimentationvalue of about 25S±2S, which operationally defines an apparent nativemolecular mass of the complex containing gp85-97 subunits.

"Sedimentation value" is defined as the value for gp85-97 extrapolatedfrom the sedimentation behavior of standards of known sedimentationvalue and molecular weight as analyzed on 5-20% sucrose gradientsdescribed in the examples. The sedimentation or S value of a nativeprotein is very reproducible under defined conditions and has been usedto estimate the molecular mass of proteins and complexes. Precisedetermination of the molecular mass of large complexes is known to bedifficult and may be influenced by the density and shape of the complexin solution. As ionizable amino and carboxyl groups are present in themolecule, gp85-97 may be obtained as an acidic or basic salt, or inneutral form. All such preparations that retain their activity whenplaced in suitable environmental conditions are included in thedefinition of proteins herein. Further, the primary amino acid sequenceof the protein may be augmented by derivatization using sugar moieties(glycosylation) or by other supplementary molecules such as lipids,phosphate, acetyl groups and the like, as well as by conjugation withsaccharides, polyethylene glycols (PEGs) and polyoxyethylene glycols(POGs). Certain aspects of such augmentation are accomplished throughpost-translational processing systems Of the producing host; other suchmodifications may be introduced in vitro. In any event, suchmodifications are included in the definition of gp85-97. It is expected,of Course, that such modifications may quantitatively or qualitativelyaffect the activity, either by enhancing or diminishing the activity ofthe protein in the various assays. Further, individual amino acidresidues in the chain may be modified by oxidation, reduction or otherderivatization, and the protein may be cleaved to obtain fragments whichretain activity. Certain modifications to the primary structure itselfby deletion, addition or substitution of the amino acids incorporatedinto the sequence during translation can be made without destroying theactivity of the protein. Such substitutions which do not destroyactivity do not remove the protein sequence from the definition and areconsidered to have substantially equivalent amino acid sequences. Inaddition N-terminal and C-terminal deletions and fusions, may be madeusing known mutagenesis methods.

It is particularly important to appreciate that native gp85-97 is aglycoprotein and that it is difficult to define solely on the basis ofits apparent molecular weight on SDS-polyacrylamide electrophoresis(SDS-PAGE). For example, glycosidase treatment of the molecule, asdescribed below, causes a decrease in the molecular weight as revealedby SDS-PAGE. It will thus be further appreciated that gp85-97 isolatedfrom different sources may exhibit different molecular weights whendetermined by SDS-PAGE, in pan as a result of variable glycosylation.

As used herein, "chromatography" is defined to include the applicationof a solution containing a mixture of compounds to an adsorbent, orother support material which is eluted, usually with a gradient or othersequential eluant. Material eluted from the support matrix is designatedthe eluate. The sequential elution is most routinely performed byisolating the support matrix in a column and passing the elutingsolution(s), which changes affinity for the support matrix, eitherstepwise or preferably by a gradient, through the matrix. It will beappreciated that encompassed within the definition of."chromatography"is the positioning of the support matrix in a filter and the sequentialadministering of eluant through the filter, or in a bitch-mode.

The phrase "hydrophobic interaction matrix" is defined to mean anadsorbent that is a hydrophobic solid such as polystyrene resin beads,rubber, silica-coated silica gel, or cross-linked agarose sufficientlysubstituted with hydrophobic functional groups to render the materialhydrophobic. Alkyl substituted agarose and aryl substituted agarose suchas, for example, phenyl or octyl agarose are representative hydrophobicmaterials. Mixtures of materials that are chromatographically separatedon a hydrophobic interaction chromatography matrix are generally firstadsorbed to the matrix in a high salt solution, and subsequentlydesorbed from the matrix by elution in a low salt solution, or ahydrophobic solvent such as a polyol.

"Anion exchange matrix" is defined to mean a solid or gel support matrixthat is charged in aqueous solutions. The support matrix may be agarosesufficiently substituted with amine functional groups to have a netcharge in aqueous solutions. The material to be adsorbed is generallybound to the anion exchange matrix in a low salt solution and isgenerally eluted from the anion exchange matrix in a high-salt eluantcontaining anions such as chloride ion which bind to the anion exchangematrix and displace the adsorbed material.

By the phrase "high-salt concentration conditions" is meant an aqueoussolution wherein an ionic substance is present to create conditions ofhigh ionic strength. Ionic strength is defined as is generallyunderstood in the art and can be calculated from the putativeconcentrations of the various ions placed in solution, modified by theiractivity coefficient High salt concentrations that are routinelyemployed are typified by solutions containing high concentrations ofammonium sulfate; however, other salts, such as sodium chloride,potassium chloride, sodium sulfate, sodium nitrate, or sodium phosphatemay also be employed.

The definition of "affinity chromatography" is understood to be similarto that of Wilchek et al., 1984, Methods in Enzymology, 104:3. "Affinitychromatography" is defined as a "method of purification based onbiological recognition". Briefly, the procedure involves coupling aligand to a solid support, and contacting the ligand with a solutioncontaining therein a ligand recognition molecule which binds to theligand. Subsequently, the ligand recognition molecule is released fromthe ligand and isolated in pure form. It will be understood that avariety of ligands can be employed in affinity chromatography asdiscussed by Wilchek, et al., and examples of these include lectins,antibodies, receptor-binding proteins and amino acids.

"Cells" or "recombinant host" or "host cells" are often usedinterchangeably as will be clear from the context. These terms includethe immediate subject cell, and, of course, the progeny thereof. It isunderstood that not all progeny are exactly identical to the parentalcell, due to chance mutations or differences in environment However,such altered progeny are included when the above terms are used.

The following relates to the antibody aspect of the invention.

"Antibody" as used herein refers to polyclonal, monoclonal andrecombinant constructs. Thus, the term includes whole immunoglobin aswell as antigen-binding fragments thereof.

The instant invention provides a description of a class of substantiallypurified molecules, hereinafter referred to as gp85-97, and of DNAsequences that encode members of the class, and materials and methodsfor identifying and isolating the same. Additionally, vectors forexpressing the DNA sequences are also shown. Members of gp85-97, oractive fragments derived therefrom, are useful as medicaments fortreating or preventing a variety of diseases.

The identification and isolation of the instant gp85-97 DNA sequenceswere made possible by the design of DNA oligonucleotide probessubstantially homologous to the DNA sequences predicted by the proteinsequences obtained from gp97. Because such probes were generated basedon a knowledge of the partial amino acid sequence of gp97, the order ofdiscussion of the invention will be: methods of assaying for gp85-97;purification of gp97; the partial amino acid sequence of gp97; cloningof the gene encoding gp85-97 using novel gp97 probes; and theidentification of gp97 DNA sequences in a cDNA library, along withsubcloning; and expression of the sequences.

I. Assay for gp85-97 Activity

One of the properties of gp85-97 is its ability to inhibit the mitogenicresponse of thymocytes stimulated with phytohaemagglutinin (PHA).Certain other mitogenic lectins, such as ConA, are not measurablyinhibited by gp85-97, suggesting significant specificity for PHA.Although PHA apparently stimulates DNA synthesis in a large number oflymphocyte populations (unlike true antigenic stimulation which causesmitogenesis of sub-populations of lymphocytes), the susceptibility of apatient's lymphocytes to PHA stimulation has been shown to correlatewith the overall immune responsiveness of the patient. Thus, this assayis widely used to study immune responsiveness.

The procedure for carrying our the phytohaemagglutinin proliferationassay (PHA/PBL) is well known to those skilled in the art. Briefly, itconsists of isolating human lymphocytes and incubating an appropriatenumber of the cells in a suitable physiological solution with anappropriate amount of PHA. Tritiated thymidine is added 48 hours laterand allowed to incubate for 24 hours before the cells are washed andcounted. Addition of various dilutions of gp85-97 prior to incubationwith PHA inhibits stimulation, resulting in less incorporation ofradioactive thymidine into DNA, and permitting an estimation of therelative concentration of gp85-97 in the solution being assayed.

A second assay (PHA/IL-1) consists of measuring gp85-97 activity basedon its capacity to inhibit IL-2 production from cell lines that arestimulated to produce IL-2 in the presence of PHA and IL-1. A variety ofcell lines are known to hive this property and the preferred cell lineis a murine T-cell line termed LBRM. IL-2 may be measured by severalassays, the preferred assay being the enumeration of viable HT-2 cellsfound after an 18-24 hour period. The HT-2 cell line is a IL-2 dependentmouse helper T-lymphocyte cell line which dies in the absence of IL-2.The assay is described by Gillis et al., 1978, J. of Immunol., 120:2027.Briefly, the proliferation of HT-2 cells in response to IL-2 is measuredby a ³ H!thymidine ( ³ H!TdR) incorporation microassay. The HT-2 cellsare washed and resuspended at 2×10⁵ /ml RPMI 1640 media containing 10%FBS. Equal volumes of cells and of serial dilutions of recombinantIL-2-containing samples are added to 96-well microtiter plates(Falcon/Becton-Dickinson Labware, Oxnard, Calif. U.S.A.). After 24hours, incubation cultures were pulsed for 5 hours with 1 μCi ³ H!TdR(specific activity, 70 Ci/mmol; New England Nuclear, Boston, Mass.,U.S.A.), harvested onto Whatman GF/C filters (Whatman LaboratoryProducts, Inc., Clifton, N.J., U.S.A.), and radioactivity determined ina liquid scintillation counter. IL-2 activity of unknown Samples ismeasured relative to a recombinant human IL-2 standard calibrated inInternational Units.

Other methods for measuring gp85-97 were based on the ability of nativeor denatured gp85-97 to bind ¹²⁵ I-labelled PHA or anti-gp85-97 antibodywhich was either ¹²⁵ I-labelled or detected by fluorescence generated byHRP-conjugated goat-anti-rabbit antibody using the ECL kit (Amersham).Approximate concentrations of gp85-97 were determined fromautoradiograms of SDS-PAGE blots or non-denatured dot-blot assays probedwith the above ligands.

II. Sources of gp85-97

A variety of biological materials are available as sources of gp85-97.Established cell lines may be utilized, and indeed are a preferredsource because of the ease with which they can be manipulated and scaledup. For some cell lines, gp85-97 is secreted and thus is present ingreatest amounts in the culture medium. For example, gp97 is preferablyisolated from SK-BR-3 cell culture media. Thus, culture medium can bethe primary source for the molecule. Additional members of gp85-97 maybe isolated from other biological sources; for example, human milk is asource of gp85. A PHA-binding protein that reacts with anti-gp85-97 Abwas also detected in supernatants from the A375 human melanoma cellline.

III. Purification of gp85-97

Preferred methods of purifying gp85-97 include various applications ofchromatography and separations based on molecular mass, such assedimentation velocity gradients and size exclusion HPLC. An example ofchromatography that effects the separation of molecular structures basedon charge differences, and that is employable in the instant invention,is anion exchange chromatography. While a variety of anionicchromatographic materials can be employed, DEAE-Sepharose, obtainablefrom Pharmacia LKB Biotechnology, Inc., is preferred.

The methods for eluting proteins from anion exchangers are generallywell documented in the literature. For example, gp85-97 can be elutedfrom DEAE using a suitably buffered salt gradient. The nature of thesalt and the steepness of the gradient can be determined empirically.Exemplary of an effective salt gradient is sodium chloride varying fromabout 0-0.8 molar.

A second chromatographic technique, particularly favored in the instantinvention, is a form of hydrophobic interaction chromatography.Hydrophobic interaction chromatography (HIC) is generally defined aschromatography which affects separation of proteins based on theirhydrophobic properties by binding to alkyl groups, attached to a solidsurface, such as Phenyl-TSK HPLC column beads. The proteins can bedifferentially eluted from the solid surface with a suitable solvent.HIC will generally be employed subsequent to the initial chromatographicstep(s), and further purifies gp85-97 by binding gp85-97 and a subset ofthe contaminating proteins to the hydrophobic matrix in the presence ofhigh salt. The contaminating proteins and gp85-97 are elutable therefromupon reduction of the salt concentration: The materials and methods forutilizing hydrophobic chromatography are described by Shaltiel S., 1984,Methods in Enzymology., 104:69. While it is apparent that there are manyhydrophobic chromatographic materials and solid supports that may beemployed to purify gp85-97, we have found that Phenyl-TSK is preferred,although phenyl Sepharose is also effective.

Another chromatographic procedure, high performance liquidchromatography, or HPLC, can be applied to enhance the purity ofgp85-97. HPLC is related to the third procedure described above in thata preferred version of HPLC as applied to the purification of gp85-97employs hydrophobic chromatographic material. This method differs fromthe preceding hydrophobic chromatographic step in at least one aspect:chromatography takes place under high pressure in the presence of anappropriate salt concentration. Examples of the types of materials andmethods that can be employed are described by Regnier, F., 1983, Methodsin Enzymology, 91:137. Preferably employed in the subject invention ischromatographic material having phenyl residues. More preferred arechromatographic materials for preparative and analytical HPLC availablefrom BioRad Corporation that are sold under the name Phenyl-TSK.

In addition to the above described chromatographic techniques, it willbe apparent that size-exclusion chromatography, which effects separationof proteins excluding structures of a predefined size from thechromatographic material employed, while the chromatographic materialretains structures of lesser size, may also be employed in the instantinvention. Chromatographic materials that are used to constructexclusion columns are widely available and sold under numeroustradenames, an example being the various Sephacryls sold by PharmaciaLKB Biotechnology, Inc. By employing the appropriate chromatographicmaterial, gp85-97 may be separated from other proteins. To obtaingp85-97 in high purity, size exclusion chromatography and/or velocitygradient centrifugation may be combined with ion exchange andhydrophobic chromatography as described above. Sucrose velocity gradientpurification is preferred, but other gradients such as glycerol may alsobe employed.

Regardless of the purification procedures chosen, and depending on thenature of the biological material that gp85-97 is purified from, it isdesirable to have present in the various purification solutions one ormore protease inhibitors, for example, EDTA, PMSF, and leupeptin.Additionally, as is known in the art, certain purification steps may beconducted at temperatures that reduce the risk of gp85-97 proteolysis.

Western blotting or PHA affinity blotting may be used to monitor thepurification of gp85-97 by subjecting preparations containing gp85-97 tosodium dodecyl sulphate polyacrylamide gel electrophoresis underreducing or nonreducing conditions (Laemmli, U., 1970, Nature,227:680-685), and blotting and probing the gels with antibody to gp85-97or with labelled PHA generally as described by Burnette, 1981, Anal.Bio. Chem., 112:195, or modifications of Burnette's method. Nativegp85-97 may also be detected by dot-blotting of undenatured protein andprobing with antibody or labelled PHA.

IV. Cloning of gp85-97

A. General Methods:

1) Cloning With Polymerase Chain Reaction

A specific nucleic acid sequence may be cloned into a vector using thepolymerase chain reaction (PCR), and primers to amplify the sequencewhich comas restriction sites on their non-complementary ends. PCR isdescribed in U.S. Pat. Nos. 4,683,195, issued Jul. 28, 1987, 4,683,202,issued Jul. 28, 1987 and 4,800,159, issued Jan. 24, 1989. In general,the synthesis/amplification of DNA sequences by PCR involves anenzymatic chain reaction that produces, in exponential quantifies, aspecific DNA sequence, provided that the termini of the sequence areknown in sufficient detail so that oligonucleotide primers can besynthesized which will hybridize to them, and that a portion of thesequence is available to initiate the chain reaction. The primers areannealed to denatured DNA, followed by extension with a suitable DNApolymerase enzyme, such as the large fragment of DNA polymerase I(Klenow), or preferably a DNA polymerase that is stable in the presenceof detergents and nucleotides, which results in newly synthesized plusand minus strands containing the target sequence. Alternatively, athermostable enzyme present in thermostable bacteria may be used. Theenzyme may be produced using DNA recombinant techniques as described inU.S. Ser. No. 063,509, filed Jul. 17, 1987. Because the newlysynthesized sequences in the PCR synthetic reaction are also templatesfor the primers, repeated cycles of denaturing, primer annealing andextension result in exponential accumulation of the region flanked bythe primers. PCR thus produces discrete nucleic acid duplexes of cDNAinserts having termini corresponding to the ends of the specific primersemployed.

Also useful is the Thermal Cycler instrument (Perkin-Elmer CetusInstruments) which has been described in European Patent Publication No.236,069, published Sep. 9, 1987 also incorporated herein by reference inits entirety.

An alternative to the use of plasmid DNAs encoding gp85-97 or fragmentsthereof as template for PCR is the use of RNA from any cell producingthese molecules as template for PCR as described in U.S. Pat. No.4,800,159. If RNA is the available starting material, the extensionproduct synthesized from one primer, when separated from its complement,can serve as a template for synthesis of the extension product of theother primer. As previously mentioned, each primer contains arestriction site near its 5' end which is the same as or different fromthe restriction site on the other primer. After sufficient amplificationhas occurred, the amplification products are treated with theappropriate restriction enzyme(s) to obtain cleaved products in arestriction digest. The desired fragment to be cloned is then isolatedand ligated into the appropriate cloning vector.

Although PCR can be performed using a variety of reaction conditions, asdescribed in the references presented above, the preferred reactionconditions are as follows. Plaques that hybridize to a particular probeare eluted into either 0.5 ml of water, or a suitably buffered solution,and 50 μl of the eluate combined with 10 μl of 10 x PCR buffer, 1.5 μlof 10 mM dNTP's, 1 μl of a first and second primer, each at aconcentration of about 20 pmoles, 0.2 μl of Taq polymerase, equivalentto 1 unit of activity. The final volume is 100 μl. PCR 10 x bufferconsists of 500 mM KCl, 200 mM Tris-HCl, pH 8.4, 25 mM MgCl₂ and 1mg/ml.

Construction of suitable vectors containing the desired gp85-97 codingsequence employs standard ligation and restriction techniques that arewell understood in the art. Isolated vectors, DNA sequences, orsynthesized oligonucleotides are cleaved, tailored, and religated in theform desired. A brief description of some of these methods is presentedhere. General cloning and molecular biology techniques are described byManiatis et el, T., et al., 1989, Molecular Cloning, Cold Spring HarborLab., Cold Spring Harbor, N.Y., volumes 1 and 2.

Site-specific DNA cleavage is performed by treating the DNA withsuitable restriction enzyme(s) under conditions that are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. See,e.g., New England Biolabs, Product Catalog. In general, about 1 μg ofplasmid or DNA sequence is cleaved by 1 unit of enzyme in about 20 μl ofbuffer solution. In the examples herein, typically, an excess ofrestriction enzyme is used to ensure complete digestion of the DNAsubstrate. Incubation times of about 1 to 2 hours at about 37° C. areworkable, although variations can be tolerated. After each incubation,protein is removed by extraction with phenol/chloroform, and may befollowed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol, followed bychromatography using a Sephadex G-S0 spin column. If desired, sizeseparation of the cleaved fragments may be performed by polyacrylamidegel or agarose gel electrophoresis using standard techniques. A generaldescription of size separations is found in Methods in Enzymology,65:499-560 (1980).

Restriction fragments may be blunt-ended by treating with a largefragment of E. coli DNA polymerase I that is known as the Klenowfragment in the presence of the four deoxynucleotide triphosphates(dNTPs) using incubation times of about 15 to 25 minutes at 20° to 25°C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl₂, 6 mM DTT and 10 mMdNTPs. After treatment with Klenow, the mixture is extracted withphenol/chloroform and ethanol precipitated. Treatment under appropriateconditions with S1 nuclease may also be used to hydrolyzesingle-stranded portions.

Ligations are generally performed in 15-30 μl volumes under thefollowing standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10mM MgCl₂, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 1-40 μM,ATP, 0.01-0.02 (Weiss) Units T4 DNA ligase at 0° C. for "sticky end"ligation, or for "blunt-end" ligations 1 mM ATP and 0.3-0.6 (Weiss)units T4 ligase at 14° C. Intermolecular "sticky end" ligations areusually performed at 33-100 μg/ml total DNA concentration. In blunt-endligations, the total DNA concentration of the ends is about 1 μM.

In vector construction employing "vector fragments", the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) in orderto remove the 5' phosphate and prevent religation of the vector. BAPdigestions are conducted at pH 8 in approximately 150 mM Tris, in thepresence of Na⁺ and Mg⁺² using about 1 unit of BAP per μg of vector at60° C. for about 1 hour. Nucleic acid fragments are recovered byextracting the preparation with phenol/chloroform, followed by ethanolprecipitation. Alternatively, religation can be prevented in vectorswhich have been double digested by additional restriction enzymedigestion of the unwanted fragments.

In the constructions set forth below, correct ligations are confirmed byfirst transforming the appropriate E. coli strain with the ligationmixture. Successful transformants are selected by resistance toampicillin, tetracycline or other antibiotics, or using other markersdepending on the mode of plasmid construction, as is understood in theart. Miniprep DNA can be prepared from the transformants by the methodof D. Ish-Howowicz et al., 1981, Nucleic Acids Res, 9:2989 and analyzedby restriction and/or sequenced by the dideoxy method of F. Sanger etal., 1977, PNAS (USA), 74:5463 as further described by Messing et al.,1981, Nucleic Acids Res., 9:309, or by the method of Maxam et al., 1980,Methods in Enzymology, 65:499.

Host strains used in cloning in M13 consist ofE. coli strains,susceptible to phage infection, such as E. coli. K12 strain DG98. TheDG98 strain was deposited with ATCC, on Jul. 13, 1984 and has AccessionNo. 1965.

Transformation is done using standard techniques appropriate to thechosen host cells. The calcium treatment employing chloride, asdescribed by S. N. Cohen, 1972, PNAS (USA), 69:2110, or the RbCl₂ methoddescribed in Maniatis et al., 1982, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, p. 254 may be used for procaryotes.Transfection of insect cells, such as for example, Sf9 cells, may beachieved using a modification of the calcium phosphate precipitationtechnique (Graham, F. L. et al., 1973, Virology, 52:456) as adapted forinsect Cells (J. P. Burand et al., 1980, Virology, 101; E. B. Casstenset al., 1980, Virology, 101:311).

B. Oligonucleotide Synthesis:

Synthetic oligonucleotides may be prepared by the triester method ofMatteucci et al., 1981, J. Am Chem. Soc., 103:3185 or using commerciallyavailable automated oligonucleotide synthesizers. Kinasing of singlestrands prior to annealing or for labeling is achieved using an excess,e.g., approximately 10 units of polynucleotide kinase to 0.1 nmolesubstrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl₂, 5 mMdithiothreitol, 1-2 mM ATP, 1.7 pmoles λ³² P-ATP (2.9 mCi/mmole), 0.1 mMspermidine, 0.1 mM EDTA.

Using the partial N-terminal and internal gp85-97 amino acid sequencesdescribed below, and known codon redundancies thereto, DNAoligonucleotides were synthesized and used as probes for probing cDNAlibraries for sequences that encode gp85-97, or as primers forconducting PCR reactions, discussed below.

C. Identification and Isolation of gp85-97 DNA Sequences:

Several procedures are available that may be suitable for identifyinggp85-97 DNA sequences. One procedure is to use the oligonucleotideprobes identified and synthesized as described above to screen cDNAlibraries. cDNA libraries can be constructed using techniques known inthe art, or can be purchased commercially.

An illustrative procedure for making a cDNA library containing gp85-97sequences may consist of isolating total cytoplasmic RNA from a suitablestarting material, and further isolating messenger RNA therefrom. Thelatter can be further fractionated into Poly (A+) messenger RNA, whichin turn is fractionated further still into Poly (A+) messenger RNAfractions containing gp85-97 messenger RNA. The appropriate gp85-97messenger RNA can then be reverse transcribed and cloned into a suitablevector to form the cDNA library.

More specifically, the starting material (i.e., tissue, cells) is washedwith phosphate buffered saline, and a non-ionic detergent, such as apolymer of ethylene oxide. For example, NP-40 is added in an amount tolyse the cellular membranes preferentially, generally about 0.3%. Nucleican then be removed by centrifugation at 1,000 x g for 10 minutes. Thepost-nuclear supernatant is added to an equal volume of TE (10 mM Tris,1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5) saturatedphenol/chloroform (1:1) containing 0.5% sodium dodecyl sulfate (SDS) and10 mM EDTA. The supernatant is re-extracted 4 times and phase separatedby centrifugation at 2,000 x g for 120 minutes. The RNA is precipitatedby adjusting the samples to 0.25M NaCl, adding 2 volumes of 100% ethanoland storing at -20° C. The RNA is then pelleted at 5,000 x g for 30minutes, washed with 70% and 100% ethanol, and dried. This representsthe total cytoplasmic RNA. Polyadenylated (Poly A+) messenger RNA (mRNA)can be obtained from the total cytoplasmic RNA by chromatography onoligo (dT) cellulose (J. Aviv et al., 1972, PNAS, 69:1408-1412). The RNAis dissolved in ETS (10 mM Tris, 1 mM EDTA, 0.5% SDS, pH7.5) at aconcentration of 2 mg/ml. This solution is heated to 65° C. for 5minutes, then quickly chilled to 4° C. After bringing the RNA solutionto room temperature, it is adjusted to 0.4M NaCl and slowly passedthrough an oligo (dT) cellulose column previously equilibrated withbinding buffer (500 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 7.5). Theflow-through is passed over the column twice more, and the column washedwith 10 volumes of binding buffer. Poly (A+) mRNA is eluted withaliquots of ETS, extracted once with TE-saturated phenol chloroform andprecipitated by the addition of NaCl to 0.2M and 2 volumes of 100%ethanol. The RNA is reprecipitated twice, washed once in 70% and then100% ethanol prior to drying. The poly (A+) mRNA can then be used toconstruct a cDNA library.

cDNA can be made from the enriched mRNA fraction using oligo (dT)priming of the poly A tails and AMV reverse transcriptase employing themethod of Okayama, H., et al., 1983, Mol. Cell Biol., 3:280,incorporated herein by reference.

Other methods of preparing cDNA libraries are, of course, well known inthe art. One method uses oligo (dT) primer, reverse transcriptase,tailing of the double stranded cDNA with poly (dG) and annealing into asuitable vector, such as pBR322 or a derivative thereof, which has beencleaved at the desired restriction site and tailed with poly (dC). Aderailed description of this alternative method is found, for example,in U.S. Ser. No. 564,224, filed Dec. 20, 1983, and assigned to the sameassignee, incorporated herein by reference. Indeed, this method ispreferred and was used to create a cDNA library from the THP-1 cell lineusing the vector pCDL-SRα296 to identify and isolate gp85-97 DNAsequences.

A preferred cDNA library would be made applying the methods describedabove and using mRNA from the human monocytic leukemia cell line, THP-1(Dr. Tsuchiga at Res. Inst. for Tuberculosis and Cancer, Tohoku Univ.Japan).

Most preferred is to construct a cDNA library using mRNA isolated frommezerin-treated THP-1 cells, and the vector pCDL SRα-296. pCDLSRα-296may be obtained from DNAX Corporation, and is described by Takebe etal., 1988, Molecular and Cellular Biology, 8(1):466; and in U.S. Pat.No. 4,695,542.

Finally, as mentioned above, cDNA libraries are commercially available,and can be purchased and used to identify and isolate the desiredgp85-97 DNA sequences. A particularly useful library is sold by Clontech(Catalog number #L H1008). It is a λ gt11 human placental cDNA librarymade from total poly (A+) messenger RNA.

V. Antibody to gp85-97

Polyclonal, monoclonal, or recombinant antibodies to gp85-97 may beproduced using various techniques. The antibody is preferably human orhumanized, although non-human antibody will perform satisfactory. Thepreparation of high-titer neutralizing polyclonal antibody can berealized by immunizing a variety of species and employing one of severaldifferent immunization regimes. The preferred method of the instantinvention is to immunize rabbits with gp85-97 prepared in completeFreund's adjuvant. A native complex containing either the 85-97 kDmolecule and/or fragments derived therefrom may be utilized asimmunogen. The animals are subsequently subjected to multiple boosts(containing about half the original amount of the appropriate molecule)in incomplete Freund's adjuvant at about 21-day intervals. About 10 daysfollowing each 21-day interval, 20-30 ml of blood is removed, the serumisolated and antibody isolated therefrom. This procedure may be carriedout for a period of several months.

Monoclonal antibody may be produced using native gp85-97 complex, itssubunit, or fragments derived therefrom as immunogen, and using theprocedures described by Kohler, G. and Milstein, C., 1975, Nature,256:495, or modifications thereof that are known in the art.

The work of Kohler and Milstein, above, involves fusing murinelymphocytes and drug selectable plasmacytomas to produce hybridomas. Asuitable plasmacytoma is Sp 2/0-Ag14 and is widely used by practitionersof this art. Subsequent to the work of Kohler and Milstein, thehybridoma technique has been applied to produce hybrid cell lines thatsecrete human monoclonal antibodies. The latter procedures are generallydescribed in Abrams, P., 1986, Methods in Enzymology, 121:107, but othermodifications are known to those skilled in the art.

Regardless of whether murine or human antibody is produced, theantibody-secreting cells are combined With the fusion partner and thecells fused with a suitable fusing agent, preferably polyethyleneglycol, and more preferably polyethylene glycol 1000. The latter isadded to a cell pellet containing the antibody-secreting cells and thefusion partner in small amounts over a short period of time accompaniedWith gentle agitation. After the addition of the fusing agent, the cellmixture is washed to remove the fusing agent and cellular debris, andthe cell mixture consisting of fused and unfused cells seeded intoappropriate cell culture chambers containing selective growth media.After a period of several weeks, hybrid cells are apparent, and may bescreened for appropriate antibody production and subcloned to ensure theavailability of a stable hybrid cell line. Cells may also be fused usingelectrofusion techniques as is known in the art.

The preferred antibody is human monoclonal antibody which can beprepared from lymphocytes sensitized with gp85-97 material, either invivo or in vitro by immortalization of antibody-producing hybrid celllines, thereby making available a permanent source of the desiredantibody, using the cell fusion techniques described above.Alternatively, sensitized lymphocytes may be immortalized by acombination of two techniques, viral transformation and cell fusion. Thepreferred combination consist of transforming antibody-secreting cellswith Epstein-Barr virus, and subsequently fusing the transformed cellsto a suitable fusion partner. Such fusion partners are known in the art,and exemplary partners may be a mouse myeloma cell line, a heteromyelomaline, or a human myeloma line, or other immortalized cell line. PCTPatent Application No. 81/00957; Schlom et al., 1980, PNAS USA, 77:6841;Croce et al., 1980, Nature, 288:488. The preferred fusion partner is amouse-human hetero-hybrid, and more preferred is the cell linedesignated F3B6. This cell line is on deposit with the American TypeCulture Collection, Accession No. HB8785. It was deposited Apr. 18,1985. The procedures for generating F3B6 are described in EuropeanPatent Application, Publication No. 174,204. Techniques applicable tothe use of Epstein-Barr virus transformation and the production ofimmortal antibody secreting cell lines are presented by Roder, J. etal., 1986, Methods in Enzymology, 121:140.

It will be apparent to those skilled in the art, and as mentioned above,while the preferred embodiment of the instant invention is neutralizinggp85-97 monoclonal antibody, such antibody(s) may be altered and stillmaintain biological activity. Thus, encompassed within the scope of theinvention is antibody modified by reduction to various size fragments,such as F(ab')₂, Fab, Fv, or the like. Also, the hybrid cell lines thatproduce the antibody may be considered to be a source of the DNA thatencodes the desired antibody, which may be isolated and transferred tocells by known genetic techniques to produce genetically engineeredantibody. An example of the latter would be the production of singlechain antibody having the antibody combining site of the hybridomasdescribed herein. Single chain antibody is described in U.S. Pat. No.4,704,692.

A second example of genetically engineered antibody is recombinant, orchimetic antibody. Methods for producing recombinant antibody are shownin U.S. Pat. No. 4,816,567, to Cabilly, et al.; Japanese patentapplication, Serial No. 84-169370, filed Aug. 15, 1984; U.S. Ser. No.644,473, filed Aug. 27, 1984; British patent application 8422238, filedon Sep. 3, 1984; Japanese patent application, No. 85-239543, filedOctober 28, 1985; U.S. Ser. No. 793,980 on Nov. 1, 1985; U.S. Ser. No.77,528, filed Jul. 24, 1987. Also, British patent application, No.867679, filed Mar. 27, 1986, describes methods for producing an alteredantibody in which at least pans of the complementary determining regions(CDRs) in the light or heavy chain variable domains have been replacedby analogous parts of CDRs from an antibody of different specificity.Using the procedures described therein it is feasible to constructrecombinant antibody having the CDR region of one species grafted ontoantibody from a second species that has its CDR region replaced.

Having described what the applicants believe their invention to be, thefollowing examples relating to gp85 and gp97 isolated from human milkand SK-BR-3 cells, respectively, are presented to illustrate theinvention and are not to be construed as limiting the scope of theinvention. For example, variation in the source, type, or methods may beemployed without departing from the scope of the present invention.

EXAMPLE 1 Assays/Purification/Characterization of SK-BR-3 gp97

I. Assays for gp85-97

A. Proliferation Assays

Activity of gp85-97 was measured using one of two PHA-stimulatedcellular assays. Human peripheral blood mononuclear cells were isolatedfrom freshly heparinized blood by density centrifugation usingFicoll-Hypaque (Pharmacia LKB Biotechnology Inc.). Mononuclear cellswere isolated from the interface, washed three times in Roswell ParkMemorial Institute 1640 (RPMI 1640) media supplemented with 1%L-glutamine, penicillin (100 units/ml), streptomycin (100 μg/ml), andthe resulting cells resuspended in media used to perform the assay.

The mononuclear cells were resuspended in a concentration of 0.67×10⁶cell/ml in assay mixture containing the following: RPMI 1640 mediacontaining 25 mM Hepes, 10% heat-inactivated fetal calf serum, 1%L-glutamine, and 2% penicillin (200 units/ml) and streptomycin (200μg/ml).

Next, 150 μl of mononuclear cell suspension containing PHA (0.25 μg/ml)were added to wells in a 96-well U-bottom tissue culture plate (Costar).This suspension contained 1×10⁵ cell/well. This mixture was added towells that contained either 50 p.1 of sample being assayed for gp85-97activity, or 50 μl of control buffer.

The 96-well plates were incubated for 3 days at 37° C. in a tissueculture incubator. Eighteen hours prior to completing the assay, thecell cultures were labelled with 0.5 uCi/well of ³ H-thymidine (2Ci/mmole). After the 18-hour radiolabelling period, cells from the96-well plates were harvested with an automatic cell harvester (Skatron,Inc., Sterling, Va.) and processed for liquid scintillation counting.Incorporation of ³ H-thymidine into DNA was measured using a LKB betaplate scintillation counter (Pharmacia LKB Biotechnology, Inc.). Thedata shown in the figures is expressed as percent inhibition ofproliferation as compared to control cells treated with mediumcontaining control buffer.

In addition to the proliferation assay described above, gp85-97 activitywas assayed by measuring the capacity of the molecule to inhibit IL-2production from LBRM-33 cells. In the presence of PHA and IL-1 this cellline secretes IL-2, which can be assayed on HT-2 cells that require IL-2for proliferation.

The assay was conducted in 96-well tissue culture plates (Falcon Corp.,3072). Chromatographic fractions sought to be tested for gp85-97activity were dialyzed into PBS and diluted into assay media consistingof RPMI 1640 supplemented with 10% fetal calf serum and 5×10⁻⁵ MB-mercaptoethanol. The final volume was 50 μl/well. Plates weresterilized by U.V. irradiation prior to assay. Next, LBRM-33 cells werediluted in the above-described assay media at 5×10⁵ per ml, and PHA-L(Sigma L-4144) added to a final concentration of 2.5 μg/ml. 100 μl/wellof LBRM-33 cells were added into the samples being tested for gp97activity. This mixture was incubated for 1 hour at 37° C. prior toadding IL-1. At the end of the 1-hour incubation period, 50 μl/well ofIL-1 was added to each well to a final concentration of 0.08 units/ml.The IL-1 was made up in assay media at a concentration 0.312 units/mi.Control samples that did not receive IL-1 received 50 μl/well of assaymedia only. The final concentration of IL-1 in the wells was 0.08units/mi. Controls include wells with LBRM-33 cells that did not receivePHA and wells that lacked both LBRM-33 cells and PHA.

Wells were then incubated at 37° C. for 18-24 hours in a tissue cultureincubator. Cells were pelleted by centrifuging the plates at 1,000 rpmfor 10 minutes, and the top 50 μl of supernatant of each well wastransferred into a new well. HT-2 cells were made up to a concentrationof 2×10⁵ cell/ml in assay media, and 50 μl of this was added per wellthat contained the 50 μl of supernatant The HT-2 cells were incubatedfor 18-24 hours at 37° C., and pulsed with 1 uCi/well of ³ H-thymidine(50 μl/well, NEN No. NET-027A) for 3-4 hours. The wells were harvestedwith an automated cell harvester and processed for liquid scintillationcounting.

B. Western and Affinity Blotting:

Western blotting, or PHA affinity blotting was used to monitor thepurification of gp85-97 and consists of subjecting preparationscontaining the molecule to sodium dodecyl sulphate (SDS) polyacrylamidegel electrophoresis under reducing, or "nonreduced" conditions (Laemmli,U., 1970, Nature., 227:680-685), and blotting and probing the blots withantibody to gp85797 or labelled PHA generally as described by Burnette,1981, Anal. Bio. Chem., 112:195, or modifications of Burnette's method.Samples were blotted onto 0.45 μm Immobilon P (Millipore), blocked withWestern buffer consisting of 3 mM KCl, 0.14M CaCl, 1.5 mM K₂ HPO₄, 8 mMNaH₂ PO₄, pH 7.4 (PBS), to which 0.02% azide, 0.1% bovine serum albumin,0.1% ovalbumin and 0.1% Tween 20 were added, probed with ¹²⁵ I-PHA-L(labelled using Pierce Iodo-Beads), washed, and counted with amulti-wire proportional detector (automated Microbiology Systems, Inc.).

In some cases the concentration of gp85-97 was also determined byserially diluting fractions, dot-blotting onto polyvinylidene difluoridepaper, probing with anti-gp85-97 Ab, and quantitative scanning ofautoradiograms standardized to dilutions of gp97. Densitometricmeasurements were performed using an Apple Macintosh II computer and16-bit Apple scanner with an Abaton Scan program. Autoradiograms of aselected dot-blot dilution were scanned, and relative gp85-97concentration was determined from the product of the size of the dottimes the average density of the dot using an Image 1.3.5 program.Absolute gp85-97 concentrations could then be determined from scans ofdilutions of known amounts of purified gp85-97 on the sameautoradiogram.

For anti-gp85-97 antibody dot blots, polyclonal rabbit antibody againstpurified native g-p97 was prepared by Berkeley Antibody Company andpurified by protein A Sepharose chromatography, followed by affinitychromatography using 97 and 70 kDa SK-BR-3 gp97 subunits (purified byhigh-performance electrophoretic chromatography) coupled to BioRadAffigel 10/15 and eluted with 0.1M glycine at pH 2. gp85-97concentration was determined by dot-blotting serially diluted samples,blocking (as above), probing with ¹²⁵ I-labelled anti-gp85-97 antibody,and performing quantitative densitometry on autoradiograms.

N-terminal gas phase sequencing of gp85-97 can be performed followingSDS-PAGE of purified material and transfer to PVDF membrane. Internalamino acid sequences can be obtained after digestion Of gp85-97 with anappropriate enzyme, for example Lys-C protease, followed by SDS-PAGE andtransfer onto PVDF membrane and sequencing using an ABI gas phasesequencer.

II. Purification of gp97

10 liters of SK-BR-3 cell culture supernatant were used as a source ofpurification of gp97. Cells were grown in serum-free and insulin-freeDMEM media for 3 days prior to collecting the media.

The conditioned medium was adjusted to contain various proteaseinhibitors, including 1 mM EDTA, 1 μg/ml leupeptin, and 200 μM PMSF.These inhibitors at these concentrations were used in all buffersthroughout the purification.

The conditioned media was concentrated 20-fold with an Amicon YM10Spiral Cartridge concentrator. The retentate was dialyzed into 25 mMTris buffer, pH 8.5, and chromatographed over a DEAE-Sepharose(Pharmacia) column having the dimensions of 5×20 cm. The protein waseluted with a 0-0.7M gradient of sodium chloride delivered at a rate of10 ml/minute with a total volume of 1.5 liters. Those fractions enrichedin gp97 were pooled and were made 0.8M in ammonium sulphate, pH 7.0,prior to initiating the next chromatographic procedure.

Three aliquots each containing a total of 150 mg of protein from theDEAE-Sepharose pooled fractions were separately applied to a BioRadpreparative phenyl-TSK HPLC column, having the dimensions 21.5×150 mm.Protein was eluted from the column at 3 ml/min with a criss-crossinggradient of decreasing ammonium sulphate (starting at 0.8M) andincreasing ethylene glycol from 0-30%. Each of the three DEAE-Sepharose,aliquots was treated in this manner and yielded a single peak of gp97following the Phenyl-TSK column. A representative example of Phenyl-TSKchromatography at this stage of the purification is shown in FIG. 1.These Phenyl-TSK column fractions were pooled, dialyzed into phosphatebuffered saline and concentrated approximately 20-fold with an Amiconstir cell using a YM10 membrane.

Next, 2 ml aliquots of the concentrated material were layered on a 37ml, 5-50% sucrose gradient buffered with phosphate-buffered saline andcentrifuged in a Beckman SW28 rotor at 24,000 rpm at 10° C. for 39hours. The bottom of the tube was punctured, and fractions collectedtherefrom. The purified gp97 was dialyzed into phosphate-buffered salinewithout protease inhibitors, filter-sterilized using a 0.45 μm acrodiscfilter (Gelman Sciences), and stored at 4° C. Starting with the 3-dayconditioned medium from SK-BR-3 cells, the purification protocol yielded3.3 mg of gp97, a fraction of which had been cleaved into 70 kD and 27kD fragments. This corresponds to a 300-fold purification with a 20%recovery.

Purified gp97 protein from an essentially identical purification wasdialyzed against PBS, concentrated by Amicon YM30 ultrafiltration to 0.5mg/ml and run on an 8% reducing SDS-PAGE that had beenpre-electrophoresed in 0.1M Tris pH 8.9 and 0.1% SDS and 0.1mMthioglycolate for 1 hour and then returned to gel running buffer(containing 0.1 mM thioglycolate) for sample separation. Protein bandswere transferred to a PVDF membrane (Pro-Blot, Applied Biosystems, Inc.)and visualized by brief Coomassie Blue staining. A predominant 70 kDband was detected and subsequently sequenced using a gas phase sequencer(Applied Biosystems). The following N-terminal sequence was obtained:VNDGDM?LAD. A 97 kD molecular weight band was also sequenced from athird, essentially identical, gp97 preparation. The following N-terminalsequence was obtained: (SEQ ID NO: 1).

A. Characterization:

The apparent native molecular weight of the purified SK-BR-3 complex wasestimated by sedimentation velocity analysis using 12 ml, 5-20% sucrose(w/v) gradients in phosphate-buffered saline (PBS) containing 2.5 mMEDTA, 0.2 mM phenylmethysulfonyl fluoride, and 2 μg/ml leupeptin.Purified SK-BK-3 gp97 (6 μg) was mixed with 0.2 mg bovine serum albuminin a total of 200 μl of PBS and centrifuged in a SW40 rotor at 28K rpmat. 15° C. for 16 hours. Fractions were collected from the bottom andassayed for marker protein by BioRad assay (read at 595 nm). A controlgradient containing 400 μg BSA and 600 μg IgM T88 in 200 μl PBS was alsorun in parallel. The gp97 peak was located by rabbit anti-gp85-97/goatanti-rabbit HRP antibody detection of 10 μl of each fraction, seriallydiluted and dot-blotted onto PVDF membrane and visualized byfluorescence. Autoradiograms were scanned to quantitate relative gp97concentration and determine the position of the peak. The sedimentationcoefficient of macromolecules has been used to determine nativemoleuclar weights. The S value of gp97 is approximately 25.

The apparent native molecular mass of the gp97 isolated from SK-BR-3cell culture supernatants was also estimated by chromatography on aSepharose 6 size exclusion HPLC column (Pharmacia) using phosphatebuffered saline, 0.5 ml/minute, as the mobile phase. FIG. 3 shows theresults. Fractions containing gp97 were identified using the ¹²⁵ I-PHAdot blot assay as described above. The apparent native molecular weightis larger than the largest standard, IgM T88, and is estimated to beover 1200 kD. The hybridoma that secretes the IgM T88 monoclonalantibody is on deposit with the American Type Culture Collection withAccession No. HB7431.

The density of purified SK-BR-3 gp97 Complex was measured by equilibriumdensity centrifugation, wherein 30 μg of gp97 was mixed into 14 ml ofphosphate-buffered saline solution containing CsCl at a density of 1.35gm/ml. This mixture was centrifuged in a Beckman SW40 rotor at 28,000rpm at 5° C. for 70 hours. The bottom of the tube was punctured, 0.5 mlfractions collected, and the density of each fraction determined byweighing aliquots of the fractions in triplicate in sealed tubes. It wasdetermined that the density of purified SK-BR-3 gp97 is approximately1.35 gm/ml.

An experiment was done to determine the sensitivity of partiallypurified gp97 to trypsin digestion. The gp97 native complex was treatedwith trypsin at a ratio of 1:10 (w/w trypsin to total protein), for 60minutes at 37° C. FIG. 4 shows a Bio-Sil SEC-250 size exclusion-HPLCchromatographic profile of the material before and after digestion.SDS-PAGE of these two preparations revealed that the semi-purified gp97was relatively protease-resistant (following limited digestion withtrypsin) into 70 kD and 27 kD fragments, while other bands were muchmore extensively degraded. The N-terminal sequence of the 70 kD band(following transfer to PVDF membrane) corresponded to that of thepurified SK-BR-3 gp97 described above.

Lectin-binding studies were carded out on gp97 (containing 7-0 kD and 27kD fragments) to partially characterize the sugar components of theglycoprotein. The reported sugar specificities of the lectins used areas follows: 1) con A (α-D-mannose and α-D-glucosamine), 2) lentil lectin(α-D-mannose), 3) wheat germ agglutinin (D-glcNAc)₂ and NeuNAc!, and 4)phytohemagglutinin, leukocyte-specific (oligosaccharides). Purified gp97(50 ng in 300 μl of PBS) was incubated 2 hours at room temperature withshaking with various lectins immobilized on various agarose beads (25 μlof beads per incubation). After removal of the beads by centrifugation,residual unbound gp97 in the supernatant was measured by theanti-gp85-97 antibody dot-blot assay described above. Only PHA-L andwheat germ agglutinin bound gp97 in significant amounts.

Similar studies were done using ¹²⁵ I-labelled PHA-L binding to gp97 runon SDS-PAGE and blotted onto PVDF paper. The effect of pretreatment withglycosidases was also studied in this system to partially characterizethe linkage of the sugars in gp97. Each glycosidase reaction was carriedout on 6 micrograms of purified gp97 that had been denatured by boilingfor 5 minutes in 0.1% sodium dodecyl sulphate containing2-mercaptoethanol in 100 mM Hepes at pH 7.0. NP40 was added to a finalconcentration of 1% (w/v), and individual 60 microliter reactions wereincubated for 18 hours at 37° C. after addition of the following unitsof enzyme: 1) 2 milliunits of neuraminidase (Boehringer MannheimBiochemicals); 2) 2 milliunits of neuraminidase, plus 1 milliunit ofO-glycan-peptide-hydrolase (Boehringer Mannheim); and 3) 0.5 milliunitsof N-glycanase (Genzyme). CaCl₂ was added to 4 mM in reactionscontaining neuraminidase. SDS-PAGE was performed in triplicate on 20microliter aliquots of each reaction, including control reactions.Products were visualized by each of the following methods; 1) CoomassieBlue staining; 2) blotting and probing with the anti-SK-BR-3 gp97antibody followed by ¹²⁵ I-protein-A; and 3) blotting and probing with¹²⁵ I-PHA.

All three detection methods revealed changes in the molecular weight ofthe molecule on SDS-PAGE following glycosidase treatment. Neuraminidasetreatment caused a small shift, implying the presence of sialic acid.O-glycan-peptide-hydrolase treatment had no additional effect,suggesting the absence of significant O-glycosylation. N-glycanasetreatment resulted in a large shift in molecular weight suggesting thepresence of significant N-linked glycosylation. ¹²⁵ I-PHA was observedto bind all antibody-reactive bands except material treated withN-glycanase, suggesting that the PHA-L binds to a N-linked sugarcomponent of gp97.

EXAMPLE 2 Cloning of the Gene Encoding Human g-p97

The gene encoding human gp85-97 was cloned using the following generalstrategy. First, SK-BR-3 gp97 which had been recovered in partiallyproteolyzed form, was denatured and reduced, and the 97 kD and 70 kDmolecules were puttied using size exclusion HPLC in 0.1% SDS. The 97 kDand 70 kD molecules were digested with Lys-C protease, and the resultingpeptides purified and sequenced. Next, based on the amino acid sequenceof one of the peptides, peptide I, degenerate oligonucleotide primerswere synthesized and used in a PCR reaction on SK-BR-3 mRNA whichyielded DNA sequences that were, in turn, used to synthesize otheroligonucleotide primers that eventually lead to the synthesis of aspecific DNA probe for screening a cDNA library to obtain thefull-length cDNA sequence that encodes SK-BR-3 gp97.

More specifically, 1 mg of partially proteolyzed SK-BR-3 g-p97 wasdenatured in 2% sodium dodecyl sulphate and reduced with 40 mMdithiothreitol. The mixture was heated to 50° C. for 10 minutes andchromatographed on a Pharmacia Superose 6 size exclusion-HPLC columnusing a mobile phase of 0.1% sodium dodecyl sulphate in 25 mM Tris (pH8.5) containing 1 mM EDTA flowing at a rate of 0.6 ml/minute. Thepurified 97 kD subunit and 70 kD fragment were separately treated with5% (w/w) Lys-C protease at 37° C. for 18 hours. To determine the extentof digestion and the peptides produced, one third of the 97 kD and 70 kDdigests were electrophoresed on reducing SDS-PAGE using a 14%acrylamide, Tricine-buffered gel. The gels were blotted using a PVDFmembrane (Pro-Blot, Applied Biosystems) and visualized by Coomassie Bluestaining. Duplicate lanes were analyzed for ¹²⁵ I-PHA binding. Theremaining two thirds of each of the digests were chromatographed byRP-HPLC using a Vydac C₄ column with acetonitrile/TFA as the mobilephase. An aliquot of each column fraction was lyophilized, and analyzedby SDS-PAGE on a 14% acrylamide Tricine-buffered gel, and the remainingprotein in each fraction was N-terminally sequenced. Lys-C proteasedigestion of both the 97 kD and 70 kD molecules produced similardigestion patterns, providing evidence that these molecules arestructurally related. Four RP-HPLC peaks were selected, and one, termedpeak I, was sequenced using an Applied Biosystems gas phase sequencerfollowing transfer to PVDF membrane. The peptide in peak I generated theN-terminal amino acid sequence set forth in (SEQ ID NO: 2).

From the amino acid sequence of peptide I, three degenerateoligonucleotide primers were synthesized that correspond, in pan, tovarious regions of the peptide. The oligonucleotides were used to primePCR reactions on SK-BR-3 poly A+mRNA. The degeneracy was decreasedsomewhat by substituting some selected wobble positions with inosine.The oligonucleotides have the following sequences: (SEQ ID NO: 3); (SEQID NO: 4); and (SEQ ID NO. 5).

The underlining marks the positions of restriction sites for HindIII for(SEQ ID 15 NO: 3) and EcoRI for (SEQ ID NO: 4) and (SEQ ID NO: 5). Therestriction sites were included in the design of the primers tofacilitate cloning the PCR products into pUC vectors.

The PCR reaction was conducted using (SEQ ID NO: 3) in combination with(SEQ ID NO: 4) or (SEQ ID NO: 5).

Briefly, PCR was performed at a final concentration of 1 x PCR buffer,50 μM dNTP's, 1 μM each of 5' and 3' primers, and 1 unit of Taqpolymerase in a total volume of 50 μl. Reaction mixtures were heated to80° C. before adding Taq polymerase. Amplification was performed usingtwo combined cycle formats. The initial 5 cycles of amplificationconsisted of: denaturation for 30 seconds at 95° C., annealing for 30seconds at 45° C. and extension for 30 seconds at 72° C. This wasfollowed by 30 cycles of amplification that consisted of denaturationfor 30 seconds at 95° C., annealing for 30 seconds at 55° C. andextension for 30 seconds at 72° C.

The predicted PCR products were anticipated to differ by about 24 basepairs, and indeed, the products obtained were approximately 97 and 121base pairs in length as judged by gel electrophoretic mobility.

The amino-terminal amino acid sequence of the SK-BR-3 gp97 wasdetermined as described above, and these dam were used to synthesizeadditional oligonucleotide primers that were used in subsequent PCRreactions with the primers (SEQ ID NO: 4), and (SEQ ID NO: 5).

The oligonucleotide primer based, in part, on the amino-terminalsequence is set forth in (SEQ ID NO: 6) and was designed as describedabove.

The PCR reactions using (SEQ ID NO: 6) and either (SEQ ID NO: 4) or (SEQID NO: 5) yielded DNA sequences of about 740 and 765 bases,respectively.

A DNA sequence was obtained from material generated in the above PCRreactions. Two additional oligonucleotide sequences were synthesized,based on this sequence, and used to probe a THP-1 cDNA library toidentify a clone that contains the sequence encoding full-length gp97.These oligonucleotide sequences are (SEQ ID NO: 7) and (SEQ D NO: 8).Their sequences are as follows: (SEQ ID NO: 7) and (SEQ D NO: 8).

EXAMPLE 3 Screening of cDNA Library for gp85-97

A cDNA library was made from mRNA isolated from THP-1 cells induced with100 ng/ml mezerein for 24 hours. The procedure consisted of isolatingthe mRNA using procedures described above and conducting first-strandcDNA synthesis by priming with oligo (dT) covalently attached tolinearized plasmid, pcDL-SRα 296.

Poly (dT) tailing was conducted using 10 x terminal deoxynucleotidyltransferase buffer (hereinafter referred to as 10 x TdT buffer) which isprepared as follows: 13.8 g cacodylic acid is added to 3.0 g Tris-basein 60 ml of water. The solution is adjusted to pH 7.6 by slow additionof solid KOH, after which the volume is increased to 88 ml with water.Subsequently, the solution is chilled to 0PC, then 2 ml of 0.1M DTT isadded, followed by the addition of 10 ml of 0.1M MnCl₂ dropwise whilethe solution is being constantly stirred. To the 20 μl of 10 x TdTbuffer is added 5.0 μl of 10 mM dTTP, and 200 μg of the Kpnendonuclease-digested pcDL-SRα 296 plasmid. An mount of water is addedto bring the solution to a total volume of 200 μl. The solution iswarmed to 37° C. for 15 minutes, and 360 units of TdT in about 3 μl isadded and incubated at 37° C. for 5 minutes.

Next, cDNA synthesis is conducted using reverse transcriptase, as isknown in the art, and (dC) tails added to the 3' hydroxyl terminus endof the newly synthesized cDNA. The (dC) tail added during the reactionto the 3' end of pcDL-SRα 296 was removed by digestion with HindIII. 100μl of the reverse transcriptase reaction contained 10 μg of poly (A+)THP-1 RNA, 20 mM Tris-HCl, pH 8.3, 2.5 mM MgCl₂, 50 mM KCl, 1 mM DTT,0.5 mM each 100 units of RNAsin 5 μg of the poly (dT) singly tailedvector-primer DNA and 100 and units of reverse transcriptase. Thereaction was run for 30 minutes at 37° C.

Finally, the oligo (dC) tailed cDNA-mRNA plasmid was circularized with aSRα promoter-linker that carded a HindIII site at one end and ahomopolymeric tail of dG at the other. The annealing reaction wasconducted under standard conditions, using an annealing solutionconsisting of 10 x annealing buffer. The 10 x annealing buffer consistsof: 0.1M Tris-HCL, pH 7.6, 1.0M NaCl, 10 mM EDTA. The mRNA strand wasreplaced by DNA by sequentially using E. coli RNase H, DNA polymerase Iand DNA ligase. The DNA was then transformed into DH5-α bacteria. (DH5-αbacteria are obtained from Bethesda Research Laboratories, ResearchProducts Division, Life Technologies, Inc., Gaithersburg, Md. 20877.Cat. No. 82585A: Max Efficiency DH5-α competent cells.)

The cDNA library obtained above may be amplified using procedures wellknown in the art, and the library may be stored by suspending cellpellets in 12.5% glycerol in LB media at -70° C.

The cDNA library was screened by plating 1.65×10⁵ transformants andusing the oligonucleotide with(SEQ D NO: 8). Thirty-one (31) colonieswere positive and were further analyzed by PCR using the primers (SEQ DNO: 7) and (SEQ ID NO: 8). Of the 31 positive colonies, 7 were found tohave approximately the predicted number of base pairs, 437. Two of the 7clones, clones 17 and 18, were chosen for further study.

Both strands of clone 18 were sequenced, using a-35S and the Sangerdideoxy method described above. Sequenase was employed as thepolymerase. The DNA sequence derived from the clone is set forth in (SEQII2) NO: 9). Residues 180-1934 encode the pro form of gp85-97. Residues234-1934 encode the mature form of gp85-97. The deduced amino acidsequence is set forth in (SEQ ID NO: 10). Amino acids 1-18 make up theputative leader sequence.

The DNA sequence of the gp85-97 gene encodes a mature protein ofsufficient size and complexity that it might be predicted to havemultiple functions. Homology searches of the DNA and protein databasesreveal the presence of a remarkably conserved domain in the N-terminalregion of human gp85-97. The first 105 amino acids of the mature gp85-97protein are over 50% identical to the extracellular terminal sequence ofthe type I human macrophage scavenger receptor. Kodama, T., et al.,1990, Nature, 343:531-535. The exact function of this domain ispresently unknown, but other domains of the scavenger receptor areapparently involved in removal of undesirable molecules such as oxidizedlow density lipoprotein, bacterial lipopolysaccharide, andsingle-stranded nucleic acid. This same domain is highly conservedbetween gp85-97 and sea urchins where it is found duplicated on thesurface of sperm and appears to function as a receptor for asperm-activating egg peptide. Dangott, L., et al., 1989, PNAS (USA),86:2128. The gp85-97 N-terminal domain is also significantly homologousto other human proteins such as the lymphocyte glycoprotein CD6. Thus,gp85-97 may exhibit functions similar to these homologous proteins, suchas involvement in responding to infectious diseases or promoting immuneresponses. The mature N-terminal sequence and apparent molecular mass ofgp85-97 are identical to that of an as yet uncloned lung carcinomaglycoprotein, the L3 antigen. Linsley et al., Biochemistry (1986)25:2978-2986.

EXAMPLE 4 Expression of SK-BR-3 gp97

Two full-length clones, clone 17 and 18, were transfected into the cellline, Cos A2. Cell line "Cos A2" a soft-agar subclone of Cos-1 cells(Gluzman, Y., 1981, Cell, 23: 175-182). Cells in log phase weretransfected using 0.05% DEAE Dextran (Sompayrac, L. M. et al., 1981,PNAS (USA), 78:7575-7578) with the appropriate plasmid. The cells wereincubated at room temperature for 1 hour and then washed to removeresidue DEAE-plasmid, and growth media was added that contained 100 uMchloroquine. After 4 hours of incubation at 37° C. in an atmosphere of5% CO₂ /95% air, the media was removed and replaced with fresh growthmedia without chloroquine. The cells were incubated in a tissue cultureincubator, and media was harvested at 24, 48, and 72 hours.

The media from the 72-hour incubations was cleared by centrifugationimmediately after harvesting and frozen for further analysis. The g-p97protein in 1 mi of the culture supernatant was immunoprecipitated bybinding to purified anti-gp85-97 antibody coupled to 40 μl of Affigel 10beads (BioRad). The bound protein was released from the pelleted, washedbeads by boiling in 20 μl of non-reducing SDS-PAGE sample buffer. Afterremoval of the beads,-samples were reduced by boiling in the presence ofadded 2-mercaptoethanol (1%) and assayed by Western blotting usingaffinity purified ¹²⁵ I-labelled anti-gp85-97 antibody as describedabove. The antibody was produced as described below. The results areshown in FIG. 5. It is apparent from the figure that gp97antibody-reactive material is produced by both Clones. It is possiblethat the observed 85 kD molecular weight differs from that of theSK-BR-3 gp97 standard because of differences in glycosylation.

EXAMPLE 5 Antibody to SK-BR-3 gp97

An immunization procedure using native SK-BR-3 gp97 (purified asdescribed above through the sucrose gradient step) was carried out overseveral months. Immunization was initiated with about 200 μg ofmaterial. About 20 days after this immunization, rabbits were boostedwith about 100 μg of the material, and bled 10 days later. Thisboosting/bleeding procedure was conducted for several months. Theinitial immunization of about 200 μg was carried out in completeFreund's adjuvant by injection into the popliteal nodes of New Zealandwhite rabbits, while the subsequent boosts were given intramuscularly inIncomplete Freund's adjuvant. At the end of the seventh month, therabbits were exsanguinated and the serum isolated. Antibody was purifiedby affinity chromatography on a protein A Sepharose column. (PharmaciaLKB Biotechnology, Inc.) For some experiments the anti-gp85-97 antibodywas further purified by ligand affinity chromatography utilizing 97 kDand 70 kD glycopeptides purified by high-performance electrophoreticchromatography and coupled to Affigel 10/15 (BioRad). Purified antibodywas eluted with glycine/HCl, pH. 2.

Studies were conducted to determine if antibody generated againstSK-BR-3 gp97 would have neutralizing activity. The antibody was testedfor its neutralizing titer against gp97 in two cell-based assays, aswell as the ¹²⁵ I-PHA dot-blot assay described above. At a dilution of1/40, the antibody was capable of a 50% neutralization of a 20 μg/mlsolution of gp97, as measured by cell-based biological assays. Sinceantibody in the absence of gp97 had no effect in the assay, it isapparent that antibody is neutralizing the effects of the gp97. Therelatively low neutralizing titer of the antibody may reflect multiplebinding sites on what is believed to be a large, multi-subunit proteincomplex. Similar dilutions of the antibody neutralized ¹²⁵ I-PHA bindingto a similar amount of immobilized native gp97.

The antibody to SK-BR-3 gp97 was tested for its ability to react withgp85-97 in various biological samples. Using Western or dot-blotanalysis the antibody reacted with semi-purified material from humanserum and purified material from human milk. Material from fresh humanserum eluted at or near the void volume on SEC-HPLC and was thus similarto material purified from SK-BR-3 cells.

EXAMPLE 6 Purification of gp85 From Human Breast Milk

400 milliliters of human milk was pooled from multiple anonymous donorsand shown to be negative for HIV and hepatitis B virus. The milk wasadjusted to contain 1 mM EDTA, 1 μg/ml leupeptin, and 200 μM PMSF. Theseinhibitors at these concentrations were used throughout thepurification.

The milk was defatted by two centrifugations at 25,000 x g for 30minutes at 4° C. and removal of the upper fat layer followed byfiltration of the aqueous layer through a glass fiber filter, andsubsequent filtration through a Gelman 50A 5 μm filter. The filtrate wasmade 0.5M in ammonium sulfate (pH 7.0) and chromatographed over a 5×20cm Phenyl-Sepharose (Pharmacia) column. The protein was eluted from thecolumn at 10 ml/min with a criss-crossing gradient of decreasingammonium sulfate and increasing ethylene glycol from 0-30%. The columnflow-through was re-chromatographed over the same column. Fractionsenriched for gp85 were pooled from both columns, dialyzed into PBS, andconcentrated 30-fold with an Amicon stir cell using a YM30 membrane.

The Amicon-concentrate material was divided into two 25 mi aliquots andeach was separately chromatographed over a S300 Sephacryl (Pharmacia)size exclusion column having the dimensions of 5×90 cm. Protein waseluted from the column at 5 ml/min. Peak fractions of gp85 were pooled,and concentrated 30-fold as above.

Next, 2 ml aliquots of the concentrated material were layered on a 37ml, 5-50% sucrose gradient buffered with PBS and centrifuged in aBeckman SW28 rotor at 27,000 rpm at 15° C. for 23 hours. The bottom ofthe tube was punctured and fractions collected therefrom.

Two-fifths of the peak fractions of gp85 was dialyzed into 50 mM Tris pH8, 150.mM NaCl containing 0.1 mM EDTA instead of 1 mM. The retentate wasmade 1 mM in MgCl₂, CaCl₂, and MnCl₂ followed by the addition of 20 mlbed volume of pre-washed Lentil Lectin Sepharose (Pharmacia). Themixture was incubated with shaking at 4° C. for 3 hours, poured into a2.6 cm diameter column and washed at ambient temperature with 50 mM TrispH 8, 150 mM NaCl minus EDTA. The column was eluted with 200 mMα-methyl-D-mannopyranoside in the same buffer. The eluted gp85 was made0.8M in ammonium sulfate, pH 7.0 and chromatographed over an analyticalPhenyl-TSK HPLC column (BioRad) having the dimensions of 7.5×75 min.Protein was eluted from the column at a flow rate of 1 ml/min under the,conditions described above. For SDS-PAGE analysis, entire fractions weredialyzed into 0.1% SDS buffered with 1 mM Tris pH 7.5, lyophilized, andresuspended in 15 μl reducing sample buffer. Bands were visualized bystaining with Coomassie Brilliant Blue. The column profile and SDS-PAGEanalysis are shown in FIG. 6. The heterogeneous distribution of apparentsubunit molecular weights from about 60 kD to over 100 kD probablyreflects heterogenecity in glycosylation.

A second aliquot of the purified preparation was fractionated onSDS-PAGE as described above and transferred to PVDF membrane asdescribed in the SK-BR-3 70 kD fragment isolation of Example 1. TheN-terminal sequence obtained using an Applied Biosystems 470A Gas-PhaseSequencer was as follows: (SEQ ID NO: 11).

The remaining three-fifths of the sucrose gradient peak fractions wassubjected to lentil-lectin chromatography as described above except thatthe column was eluted at 4° C. followed by an identical elution atambient temperature, which released additional bound protein. The gp85that eluted at 4° C. was chromatographed using analytical Phenyl-TSKHPLC as above. Fractions enriched for gp85 were pooled, dialyzed intoPBS, concentrated 10-fold and stored at -20° C. The gp85 that eluted atambient temperature was concentrated 75-fold using an Amicon YM30membrane, and the retentate was chromatographed over a PharmaciaSuperose 6 size-exclusion FPLC column. The protein was eluted from thecolumn at 0.5 ml/min with a mobile phase of PBS. Peak fractions of gp85were pooled, concentrated 2-fold, filter-sterilized, and stored at 4° C.Starting with defatted, filtered human milk, the purification protocolyielded 400 μg of gp85 purified 1300-fold with about 6% recovery.

In addition to the characterization of gp85 described above, thematerial purified by Superose 6 chromatography was analyzed by sucrosegradient sedimentation velocity as described in Example 1. Thesedimentation value of material that reacted with anti-gp85-97 antibodyin a dot-blot assay was approximately 25. Additional reactive materialwas detected with a sedimentation value of over 30.

EXAMPLE 7 Growth Inhibition of Breast Cancer Cells

Neutralizing antibody obtained as described in Example 5 was tested forcancer cell growth-inhibitory activity. The assay consisted ofdetermining the effect dilutions of a protein-A-purified anti-gp85-97antibody on cell growth as a function of thymidine incorporation. Thehuman breast tumor cell line, SK-BR-3 was tested under conditions ofminimal stimulation in nutrient-deficient medium (RPMI with limitingfetal calf serum). An anti-GAP polyclonal antibody (Halenbeck, R. etal., 1990, J. Biol. Chem., 265:21922-21928) was used as a controlfollowing an identical protein A purification.

The thymidine assay was conducted as follows: SK-BR-3 cell proliferationwas measured by adding 2 μCi/per well of ³ H-thymidine and incubatingthe cells for 4 hours at 37° C., after which the cells were washed,harvested, and counted using standards. The assay was conducted twice,with all samples being assayed in quintuplicate. The results are shownin Table 1.

                  TABLE 1                                                         ______________________________________                                        Treatment                 % of Control                                        ______________________________________                                        Experiment 1* (Control = 3156 CPM)                                            Control (Media)           100 +/- 12                                          Purified SK-BR-3 gp97 1:10 (0.05 mg/ml)                                                                 104 +/- 3                                           Purified SK-BR-3 gp97 1:40                                                                              105 +/- 5                                           Purified Anti-gp85-97 Antibody 1:10 (0.1 mg/ml)                                                          32 +/- 3                                           Antibody 1:40              40 +/- 3                                           Purified Control Antibody 1:10 (0.1 mg/ml)                                                               97 +/- 8                                           Purified Control Antibody 1:40                                                                          155 +/- 5                                           Experiment 2* (Control = 2009 CPM)                                            Control                   100 +/- 8                                           gp85-97 Antibody 1:10      43 +/- 4                                           gp85-97 Antibody 1:40      51 +/- 11                                          ______________________________________                                         *All samples assayed in quintuplicate                                    

In the first experiment, relative to the controls, which consisted ofmedia without antibody or the antigen used to generate the antibody,gp97, there was a 68% and 60% reduction in the mount of ³ H-thymidineuptake at antibody dilutions of 1:10 and 1:40, respectively. A controlantibody was shown to have essentially no effect or a stimulatory effecton ³ H-thymidine uptake.

In the second experiment, there was a 57% and 49% reduction in theamount of H-thymidine uptake at antibody dilutions of 1:10 and 1:40,respectively.

EXAMPLE 8 Detection of gp85-97 in Human Fluids

Assorted human fluids were immunoprecipitated with anti-gp85-97 antibody(as prepared above) and subjected to Western analysis (FIG. 7). Lanes1-7, respectively, are: buffer control; semen, 0.5 ml; breast milk, 0.05ml; plasma, 1.0 ml; saliva, 0.5 ml; tears, 0.2 ml; and urine, 1.0 ml.Samples were adjusted to 0.1 mM PMSF and 2 μg/ml leupeptin in a finalvolume of 1 ml of PBS. These solutions were immunoprecipitated andsubjected to non-reducing SDS-PAGE and Western analysis usingaffinity-purified ¹²⁵ I-labelled anti-gp85-97 antibody as described inExample 1-B. Immunoreactive material varied somewhat in size andcomplexity, but generally contained two bands between 60 and 100 kDa Mr.The fact that the immunoreactive material in breast milk was purifiedusing the same antibody for assay detection and had the N-terminalsequence of gp85-97 (see above) argues that immunoreactive bands ofsimilar size in other human fluids also represent gp85-97.

EXAMPLE 9 Co-Precipitation of gp85-97

HT-29 cells (ATCC HTB 38) were grown, lysed with 0.5% Triton X-100,centrifuged to remove cells and debris, immunoprecipitated usinganti-Mac-2 monoclonal antibody (M3/38, Boehringer Mannheim) and proteinG Sepharose (Pharmacia), essentially as previously described (Rosenberget al., J. Biol. Chem. (1991) 266:18731-18736). Immunoprecipitationswere also performed using purified polyclonal rabbit antibody togp85-97, immobilized on Affigel 10. HL-60 cells (ATCC CCL 240) weredifferentiated with 80 mM phorbol 12-myristate 13-acetate for 72 hoursand lysed as above.

Immunoprecipitates were analyzed by non-reducing or reducing (lanesprobed with Mac-2 antibody) SDS-PAGE and Western analysis with ¹²⁵I-labelled antibodies (Probe Ab) as shown in FIG. 8. Samples marked(+lac) and (+man) received 0.25M lactose or mannose, respectively, priorto precipitation. The first lane in each panel shows background fromimmunoprecipitation of lysis buffer. A) Lysate of human colon carcinoma,HT-29 (1.5 mg/lane). B) Purified gp97 (5 μg) mixed with lysate of humanpromyelocytic leukemia, HL-60 (0.8 mg/lane). FIG. 8 shows that gp85-97is bound by the human Mac-2 lectin via its lactose-dissociable,carbohydrate binding properties.

    ______________________________________                                        Deposition of Cultures                                                        Vector       Deposit Date                                                                             ATCC Accession No.                                    ______________________________________                                        pSKBR3-gp97  April 30, 1991                                                                           68608                                                 ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ValAsnAspGlyAspMetArgLeuAlaAspGlyGly                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LeuAlaSerAlaTyrGlyAlaArgGlnLeuGlnGlyTyrLeuAlaSer                              151015                                                                        LeuPheAlaIleLeuLeuProGlnAspProSerPheGlnMetSerLeu                              202530                                                                        AspLeuTyrAlaTyrAla                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- difference                                          (B) LOCATION: replace(11..12, "")                                             (D) OTHER INFORMATION: /note= "N = INOSINE"                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- difference                                          (B) LOCATION: replace(17..18, "")                                             (D) OTHER INFORMATION: /note= "N = INOSINE"                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- difference                                          (B) LOCATION: replace(20..21, "")                                             (D) OTHER INFORMATION: /note= "N = INOSINE"                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- difference                                          (B) LOCATION: replace(23..24, "")                                             (D) OTHER INFORMATION: /note= "N = INOSINE"                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CAAGCTTGGCNTAYGGNGCNMGNCA25                                                   (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- difference                                          (B) LOCATION: replace(18..19, "")                                             (D) OTHER INFORMATION: /note= "N = INOSINE"                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- difference                                          (B) LOCATION: replace(21..22, "")                                             (D) OTHER INFORMATION: /note= "N = INOSINE"                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GGAATTCCCATYTGRAANSWNGGRTC26                                                  (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- difference                                          (B) LOCATION: replace(14..15, "")                                             (D) OTHER INFORMATION: /note= "N = INOSINE"                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- difference                                          (B) LOCATION: replace(20..21, "")                                             (D) OTHER INFORMATION: /note= "N = INOSINE"                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GGAATTCCGCRTANGCRTANARRTC25                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- difference                                          (B) LOCATION: replace(13..14, "")                                             (D) OTHER INFORMATION: /note= "N = INOSINE"                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- difference                                          (B) LOCATION: replace(22..23, "")                                             (D) OTHER INFORMATION: /note= "N = INOSINE"                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGGCAAGCTTGTNAAYGAYGGNGAYATG28                                                (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GAGAACGCCACCCAGGCTC19                                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AGAAGTACCTGAGAAGGTCC20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2285 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AATCGAAAGTAGACTCTTTTCTGAAGCATTTCCTGGGATCAGCCTGACCACGCTCCATAC60                TGGGAGAGGCTTCTGGGTCAAAGGACCAGTCTGCAGAGGGATCCTGTGGCTGGAAGCGAG120               GAGGCTCCACACGGCCGTTGCAGCTACCGCAGCCAGGATCTGGGCATCCAGGCACGGCCA180               TGACCCCTCCGAGGCTCTTCTGGGTGTGGCTGCTGGTTGCAGGAACCCAAGGCGTGAATG240               ATGGTGACATGCGGCTGGCCGATGGGGGCGCCACCAACCAGGGCCGCGTGGAGATCTTCT300               ACAGAGGCCAGTGGGGCACTGTGTGTGACAACCTGTGGGACCTGACTGATGCCAGCGTCG360               TCTGCCGGGCCCTGGGCTTCGAGAACGCCACCCAGGCTCTGGGCAGAGCTGCCTTCGGGC420               AAGGATCAGGCCCCATCATGCTGGACGAGGTCCAGTGCACGGGAACCGAGGCCTCACTGG480               CCGACTGCAAGTCCCTGGGCTGGCTGAAGAGCAACTGCAGGCACGAGAGAGACGCTGGTG540               TGGTCTGCACCAATGAAACCAGGAGCACCCACACCCTGGACCTCTCCAGGGAGCTCTCGG600               AGGCCCTTGGCCAGATCTTTGACAGCCAGCGGGGCTGCGACCTGTCCATCAGCGTGAATG660               TGCAGGGCGAGGACGCCCTGGGCTTCTGTGGCCACACGGTCATCCTGACTGCCAACCTGG720               AGGCCCAGGCCCTGTGGAAGGAGCCGGGCAGCAATGTCACCATGAGTGTGGATGCTGAGT780               GTGTGCCCATGGTCAGGGACCTTCTCAGGTACTTCTACTCCCGAAGGATTGACATCACCC840               TGTCGTCAGTCAAGTGCTTCCACAAGCTGGCCTCTGCCTATGGGGCCAGGCAGCTGCAGG900               GCTACTGCGCAAGCCTCTTTGCCATCCTCCTCCCCCAGGACCCCTCGTTCCAGATGCCCC960               TGGACCTGTATGCCTATGCAGTGGCCACAGGGGACGCCCTGCTGGAGAAGCTCTGCCTAC1020              AGTTCCTGGCCTGGAACTTCGAGGCCTTGACGCAGGCCGAGGCCTGGCCCAGTGTCCCCA1080              CAGACCTGCTCCAACTGCTGCTGCCCAGGAGCGACCTGGCGGTGCCCAGCGAGCTGGCCC1140              TACTGAAGGCCGTGGACACCTGGAGCTGGGGGGAGCGTGCCTCCCATGAGGAGGTGGAGG1200              GCTTGGTGGAGAAGATCCGCTTCCCCATGATGCTCCCTGAGGAGCTCTTTGAGCTGCAGT1260              TCAACCTGTCCCTGTACTGGAGCCACGAGGCCCTGTTCCAGAAGAAGACTCTGCAGGCCC1320              TGGAATTCCACACTGTGCCCTTCCAGTTGCTGGCCCGGTACAAAGGCCTGAACCTCACCG1380              AGGATACCTACAAGCCCCGGATTTACACCTCGCCCACCTGGAGTGCCTTTGTGACAGACA1440              GTTCCTGGAGTGCACGGAAGTCACAACTGGTCTATCAGTCCAGACGGGGGCCTTTGGTCA1500              AATATTCTTCTGATTACTTCCAAGCCCCCTCTGACTACAGATACTACCCCTACCAGTCCT1560              TCCAGACTCCACAACACCCCAGCTTCCTCTTCCAGGACAAGAGGGTGTCCTGGTCCCTGG1620              TCTACCTCCCCACCATCCAGAGCTGCTGGAACTACGGCTTCTCCTGCTCCTCGGACGAGC1680              TCCCTGTCCTGGGCCTCACCAAGTCTGGCGGCTCAGATCGCACCATTGCCTACGAAAACA1740              AAGCCCTGATGCTCTGCGAAGGGCTCTTCGTGGCAGACGTCACCGATTTCGAGGGCTGGA1800              AGGCTGCGATTCCCAGTGCCCTGGACACCAACAGCTCGAAGAGCACCTCCTCCTTCCCCT1860              GCCCGGCAGGGCACTTCAACGGCTTCCGCACGGTCATCCGCCCCTTCTACCTGACCAACT1920              CCTCAGGTGTGGACTAGACGGCGTGGCCCAAGGGTGGTGAGAACCGGAGAACCCCAGGAC1980              GCCCTCACTGCAGGCTCCCCTCCTCGGCTTCCTTCCTCTCTGCAATGACCTTCAACAACC2040              GGCCACCAGATGTCGCCCTACTCACCTGAGCGCTCAGCTTCAAGAAATTACTGGAAGGCT2100              TCCACTAGGGTCCACCAGGAGTTCTCCCACCACCTCACCAGTTTCCAGGTGGTAAGCACC2160              AGGACGCCCTCGAGGTTGCTCTGGGATCCCCCCACAGCCCCTGGTCAGTCTGCCCTTGTC2220              ACTGGTCTGAGGTCATTAAAATTACATTGAGGTTCCTAAAAAAAAAAAAAAAAAAAAAAA2280              AAAAA2285                                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 585 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetThrProProArgLeuPheTrpValTrpLeuLeuValAlaGlyThr                              151015                                                                        GlnGlyValAsnAspGlyAspMetArgLeuAlaAspGlyGlyAlaThr                              202530                                                                        AsnGlnGlyArgValGluIlePheTyrArgGlyGlnTrpGlyThrVal                              354045                                                                        CysAspAsnLeuTrpAspLeuThrAspAlaSerValValCysArgAla                              505560                                                                        LeuGlyPheGluAsnAlaThrGlnAlaLeuGlyArgAlaAlaPheGly                              65707580                                                                      GlnGlySerGlyProIleMetLeuAspGluValGlnCysThrGlyThr                              859095                                                                        GluAlaSerLeuAlaAspCysLysSerLeuGlyTrpLeuLysSerAsn                              100105110                                                                     CysArgHisGluArgAspAlaGlyValValCysThrAsnGluThrArg                              115120125                                                                     SerThrHisThrLeuAspLeuSerArgGluLeuSerGluAlaLeuGly                              130135140                                                                     GlnIlePheAspSerGlnArgGlyCysAspLeuSerIleSerValAsn                              145150155160                                                                  ValGlnGlyGluAspAlaLeuGlyPheCysGlyHisThrValIleLeu                              165170175                                                                     ThrAlaAsnLeuGluAlaGlnAlaLeuTrpLysGluProGlySerAsn                              180185190                                                                     ValThrMetSerValAspAlaGluCysValProMetValArgAspLeu                              195200205                                                                     LeuArgTyrPheTyrSerArgArgIleAspIleThrLeuSerSerVal                              210215220                                                                     LysCysPheHisLysLeuAlaSerAlaTyrGlyAlaArgGlnLeuGln                              225230235240                                                                  GlyTyrCysAlaSerLeuPheAlaIleLeuLeuProGlnAspProSer                              245250255                                                                     PheGlnMetProLeuAspLeuTyrAlaTyrAlaValAlaThrGlyAsp                              260265270                                                                     AlaLeuLeuGluLysLeuCysLeuGlnPheLeuAlaTrpAsnPheGlu                              275280285                                                                     AlaLeuThrGlnAlaGluAlaTrpProSerValProThrAspLeuLeu                              290295300                                                                     GlnLeuLeuLeuProArgSerAspLeuAlaValProSerGluLeuAla                              305310315320                                                                  LeuLeuLysAlaValAspThrTrpSerTrpGlyGluArgAlaSerHis                              325330335                                                                     GluGluValGluGlyLeuValGluLysIleArgPheProMetMetLeu                              340345350                                                                     ProGluGluLeuPheGluLeuGlnPheAsnLeuSerLeuTyrTrpSer                              355360365                                                                     HisGluAlaLeuPheGlnLysLysThrLeuGlnAlaLeuGluPheHis                              370375380                                                                     ThrValProPheGlnLeuLeuAlaArgTyrLysGlyLeuAsnLeuThr                              385390395400                                                                  GluAspThrTyrLysProArgIleTyrThrSerProThrTrpSerAla                              405410415                                                                     PheValThrAspSerSerTrpSerAlaArgLysSerGlnLeuValTyr                              420425430                                                                     GlnSerArgArgGlyProLeuValLysTyrSerSerAspTyrPheGln                              435440445                                                                     AlaProSerAspTyrArgTyrTyrProTyrGlnSerPheGlnThrPro                              450455460                                                                     GlnHisProSerPheLeuPheGlnAspLysArgValSerTrpSerLeu                              465470475480                                                                  ValTyrLeuProThrIleGlnSerCysTrpAsnTyrGlyPheSerCys                              485490495                                                                     SerSerAspGluLeuProValLeuGlyLeuThrLysSerGlyGlySer                              500505510                                                                     AspArgThrIleAlaTyrGluAsnLysAlaLeuMetLeuCysGluGly                              515520525                                                                     LeuPheValAlaAspValThrAspPheGluGlyTrpLysAlaAlaIle                              530535540                                                                     ProSerAlaLeuAspThrAsnSerSerLysSerThrSerSerPhePro                              545550555560                                                                  CysProAlaGlyHisPheAsnGlyPheArgThrValIleArgProPhe                              565570575                                                                     TyrLeuThrAsnSerSerGlyValAsp                                                   580585                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ValAsnAspGlyAspMetArgLeuAlaAspGlyGlyAlaThrAsnGln                              151015                                                                        GlyArgValGluIlePheTyr                                                         20                                                                            __________________________________________________________________________

The present invention has been described with reference to specificembodiments. However, this application is intended to cover thosechanges and substitutions which may be made by those skilled in the artwithout departing from the spirit and the scope of the appended claims.

We claim:
 1. A method of measuring the concentration of glycoproteincomplexes in a sample of human milk, said glycoprotein complexescomprising a plurality of glycoprotein subunits, wherein saidglycoprotein complexes have (i) an apparent molecular weight of over1200 kD and a sucrose velocity gradient sedimentation value of about25S, and (ii) the ability to interfere with PHA-dependent activation oflymphocytes, and wherein said glycoprotein subunits are about 85-97 kDon reducing SDS polyacrylamide gel electrophoresis, said methodcomprising the steps of:a) contacting a sample of human milk with anantibody which binds said gp85-97 subunits to form a mixture; and b)determining the concentration of said gp85-97 subunits in said mixture.2. The method of claim 1 wherein said antibody to said glycoproteinsubunits is obtained using the protein product of SK-BR-3 gp97 as animmunogen.